The background for and disclosure of the invention are explained with reference to the following patents and publications each of which is hereby incorporated by reference as if set forth in full herein:
(1) Milofsky, R. E. and E. S. Yeung, "Native Fluorescence Detection of Nucleic Acids and DNA Restriction Fragments in Capillary Electrophoresis", Anal. Chem., vol. 65, (1993): pp. 153-157; PA0 (2) Chi, Z., X. G. Chen, J. S. W. Holtz and S. A. Asher, "UV Resonance Raman-Selective Amide Vibrational Enhancement: Quantitative Methodology for Determining Protein Secondary Structure", Biochemistry, Vol. 37, (1988): pp. 2854-2864; PA0 (3) Thomas, G. J., Spectroscopy of Biological Systems, Ed. Clark, R. J. and R. E. Hester, John Wiley (1986); PA0 (4) Cho, N., Song, S., and S. A. Asher, "UV Resonance Raman and Excited-State Relaxation Rate Studies of Hemoglobin", Biochemistry, Vol. 33, (1994): pp. 5932-5941; PA0 (5) Cho, N., and S. A. Asher, "UV Resonance Raman and Absorption Studies of Angiotensin II Conformation in Lipid Environments", Biospectroscopy, Vol. 2, (1996): pp. 71-82; PA0 (6) Chronister, E. L., Corcoran, R. C. Song, L., and El-Sayed, M., Proc. Nat'l Acad. Sci. (USA), Vol. 83, (1986): pp. 8580-8583; PA0 (7) Barry, B., and R. A. Mathies, Biochemistry, Vol. 26, (1987): pp. 59-64; PA0 (8) Asher, S. A., Methods in Enzuymology, Vol 76, (1981): pp. 371-383; PA0 (9) Spiro, R. G., Biological Applications of Raman Spectroscopy: Vol II, John Wiley (1987); PA0 (10) Asher, S. A., Ann. Rev. Phys. Chem., Vol. 39, (1988): pp. 537-542; PA0 (11) Asher, S. A., Johnson, C. R. and J. Murtaugh, Rev. Sci. lnstr. Vol. 54, (1983): pp. 1657-1659; PA0 (12) Asher, S. A., Anal. Chem., Vol. 65, No.4, (Feb.15, 1993): pp. 201-210. PA0 (13) Gerstenberger, et al., "Hollow Cathode Metal Ion Lasers", IEEE J. Quantum Elect., vol. QE-16, No. 8, (August 1980): pp. 820-834; PA0 (14) U.S. Pat. No. 4,641,313, entitled "Room Temperature Metal Vapour Laser", to Tobin; PA0 (15) McNeil, et al., "Ultraviolet Laser Action From Cu ll in the 2500-A Region", App. Phys. Letters, vol. 28, No.4, (Feb. 15, 1976): pp. 207-209; PA0 (16) Warner, et al., "Metal-Vapor Production by Sputtering in a Hollow-Cathode Discharge: Theory and Experiment", J. App. Phys., vol. 50, No 9, (September 1979): pp. 5694-5703; PA0 (17) Solanki, et al., "Multiwatt Operation of Cull and Agll Hollow Cathode Lasers", IEEE J. Quant Elect, vol. QE-16, No.12, (December 1980): pp. 1292-1294. PA0 (18) Arslanbekov, et al., "Self-consistent Model of High Current Density Segmented Hollow Cathode Discharges", J. App. Phys., vol. 81, No 2, (January 1997): pp. 1-7; PA0 (19) U.S. Pat. No. 5,311,529, entitled "Liquid Stabilized Internal Mirror Lasers", to Hug; and PA0 (20) U.S. Pat. No. 4,953,176, entitled "Angular Optical Cavity Alignment Adjustment Utilizing Variable Distribution Cooling", to Ekstrand.
Existing lasers which emit in the deep ultraviolet between 200 nm and 300 nm have serious limitations in one or more of the following: (1) the selection of emission wavelengths, (2) average or instantaneous output power, (3) power consumption, (4) reliability, (5) size, (6) weight, and (7) cost. Because laser sources without these limitations have never been developed and commercialized, a wide range of commercial analytical instrumentation that could benefit from such sources have never been enabled.
Capillary electrophoresis, high performance liquid chromatography, laser-induced fluorescence, fluorescence microscopy, and Raman spectroscopy are emerging as powerful analytical tools for a wide range of biological and chemical research. In addition, these instrumental techniques are being increasing used in commercial applications such as product inspection during the manufacture of pharmaceutical and medical products, manufactured food products and other chemical products.
Capillary electrophoresis (CE) allows rapid separation of complex chemical and biochemical mixtures. Laser induced fluorescence (LIF) allows the sensitive detection of analytes. Raman spectroscopy (RS) allows a high level of chemical specificity. The sensitivity and selectivity of these analytical instruments are today considerably enhanced when combined with a laser, which emits in the deep UV between 200 nm and 300 nm. The principle limitation to widespread commercial use of these systems is lack of commercially suitable UV lasers, particularly associated with limitations in emission wavelengths, duty cycle, size, power consumption, complexity, cost and reliability of existing lasers. A need exists in these fields for improved laser systems, particularly in the deep UV, that overcome these disadvantages either singly or in combination.
Capillary electrophoresis continues to evolve as a powerful analytical method for separation and analysis of complex chemical analytes. A major direction of development in CE is toward smaller capillaries, allowing faster separations. As capillary diameters decrease below 50 microns and head toward 20 or even 10 microns, the problem of providing enough light to excite fluorescence in a sample being examined determines the detection limit. This is discussed in reference (1) by Milofsky and Yeung, 1993. When capillary diameters were larger, deuterium lamps were employed to excite native fluorescence in biomolecules. However, as capillary diameters decrease, deuterium lamps no longer have sufficient source radiance at the desirable deep UV wavelengths to enable them to be employed. Lasers have been recognized as the solution to this problem. However, because lasers of reasonable cost and size only emit in the visible or near IR, fluorescent dye derivatization techniques were developed to enable the use of these lasers for detection. Derivatization with fluorescent labels limits the types of molecules which can be studied, reduces CE's ability to find unexpected analytes in complex systems, may perturb the very cellular chemistry being studied, and can reduce overall sensitivity. A sensitive native CE/LIF detection method for nucleic acids and DNA restriction fragments has already been demonstrated as described in reference (1) by Milofsky and Yeung in 1993. Both the 275.4 nm line of an argon ion laser and the 248 nm line from a waveguide KrF laser were able to excite native fluorescence in the nucleic acids with a few mW of laser power. Detection limits for guanosine and adenosine monophosphate of 1.5.times.10.sup.-8 and 5.times.10.sup.-8 M, respectively, were as much as three orders of magnitude lower than UV fluorescent tag detection. However, the complexity and cost of the laser employed severely restrict the general utility of this technique. A need exists in this field for improved laser systems with reduced complexity and/or cost to make practical the above noted applications.
Raman spectroscopy has been demonstrated as a uniquely important technique for analyzing biological structure and function. Traditional Raman spectroscopy has been used to study a wide range of biological molecules such as protein secondary structure, nucleic acid folding and membrane phase transitions as described in reference (2) by Chi, et al., 1988. Most of this work has examined purified chemical systems, such as polymers, proteins, and nucleic acid systems, but a number of studies have probed complicated systems such as industrial and environmental samples, as well as DNA structure in whole viruses as described in reference (3) by Thomas, 1986.
The aromatic ring structures of tyrosine, tryptophane, and phenylalanine offer excellent LIF and UV resonance Raman (UVRR) cross-sections. The abundance of these three targets in the vast majority of proteins has made possible such investigations as the determination of protein acid denaturation using UVRR, characterization of excited-state relaxation rates in hemoglobin as described in reference (4) by Cho, et al., 1994, and elucidation of the secondary structure of angiotensin II as described in reference (5) by Cho, et al., 1996.
Resonance Raman excitation results in scattering cross-sections that are enhanced by as much as eight orders of magnitude over normal Raman spectroscopy. Resonance Raman sensitivity is comparable to that of fluorescence. Selectivity can be greater than fluorescence because Raman spectra have higher information content. Narrow Raman emission bands carry a great deal more information on molecular structure, in contrast to broadband fluorescence emission. It also allows selective study of specific chromophoric segments of a macromolecule. Visible wavelength resonance Raman spectroscopy has been uniquely incisive in the development of the understanding of energy transduction in rhodopsin and bacteriorhodopsin as described in reference (6) by Chronister, et al., 1986 and in reference (7) by Barry et al., 1987. It has also been incisive in structural and dynamical studies of numerous heme proteins such as hemoglobin and cytochrome oxidase as described in reference (8) by Asher, 1981 and in reference (9) by Spiro, 1987. However, detection of the Raman signal is usually complicated by the presence of background fluorescence from not only the molecules of interest but also from solvents and impurities.
More than ten years ago, instrumentation was developed which allowed excitation in the UV absorbing bands of molecules as described in references (10) by Asher, 1988 and in reference (11) by Asher et al., 1983. These fundamental studies have shown that unique information is available from UV resonance Raman studies of macromolecular structure. In addition, it has been shown that the ubiquitous fluorescence, which is a major impediment for visible wavelength Raman studies, does not occur for UV spectral studies below 260 nm. This is because at these high energies the excited state of most molecules in a condensed phase relaxes by means of fast radiationless processes before it has time to fluoresce as described in reference (12) by Asher, 1993.
As described above, deep UV laser radiation is useful for a wide range of commercially valuable applications. A key feature impeding the commercial development of these applications is the lack of availability of a deep UV laser of suitable cost, size, weight, power consumption and optical output properties. Thus, a need exists in these arts for a laser device that fulfills one or more of these deficiencies.
Metal ions such as those of copper, silver, and gold can provide a rich array of possible laser emission wavelengths as described in reference (13) by Gerstenberger, 1980. The historical difficulty in developing useful metal ion lasers has been associated with the method of generating an adequate metal vapor density within the gain region of the laser. Direct vaporization by evaporation or sublimation requires very high temperatures, typically about 1500.degree. C. for copper. Operation of lasers at these temperatures requires very high power consumption and is a major source of unreliability. In addition, in positive column discharge configurations of these lasers, insufficient population of high-energy states of copper or gold is developed to enable output at the deep ultraviolet emission lines.
A method of providing adequate metal vapor densities at lower operating temperature is through the use of volatile compounds of the metal such as a metal halide as described in reference (14) by Tobin. These types of metal ion lasers still require substantial heating of the volatile metal compound, to temperatures near 300.degree. C. instead of 1500.degree. C. However, in addition, there is a further limitation of these lasers due to the limited range of metals that can be combined into these suitable compounds. A further limitation of these lasers is that the self-terminating transitions described by Tobin only operate with very short pulse widths, making them undesirable for many biological applications.
One way to avoid these limitations is by the use of sputtering to achieve the desired metal ion densities. Sputtering metal ion hollow cathode lasers have been demonstrated in several laboratories starting about 1976, as described in reference (15) by McNeil, (16) by Warner, (17) by Solanki and up to the present time as described in reference (18) by Arslanbekov, et al., 1997. An advantage of the sputtering method of providing metal vapor is that this can be done at room temperature, thus avoiding the power requirements and warm-up time associated with the other metal vapor lasers noted above. These lasers have demonstrated the ability to provide emission over a wide range of wavelengths from about 200 nm in the deep UV to nearly 2000 nm in the middle infrared. Threshold for lasing varies considerably from laser line to laser line, but typically ranges from about 2A to 40A at 250V to 500V. Thus the input power to achieve threshold varies from about 500W to 10,000W.
Sputtering metal ion hollow cathode lasers described in the literature have several problems that limited their commercial use. These limitations are summarized as: too costly to manufacture both the laser plasma tube and power supply, too large, too fragile, poor lifetime and overall reliability, limited variety of laser emission lines, and limited variety of laser output performance characteristics.
Hollow cathode sputtering metal ion lasers used laser plasma tubes that were sealed on each end either with Brewster angle windows or laser mirrors. For tubes sealed with Brewster angle windows, the laser mirrors were mounted external to the laser tube. When laser mirrors were used to seal the ends of the laser tube, the critical reflecting surfaces of the mirrors were internal to the hermetic envelope of the laser plasma tube. In both cases, the structure used to maintain laser mirrors in alignment with respect to each other and with respect to the laser tube was external to the laser tube. This external structure is referred to as the external resonator structure.
In addition prior hollow cathode sputtering metal ion lasers utilized bulky, expensive, unreliable, and fragile designs for cathodes, cathode supports, and other tube design elements which made these lasers susceptible to arcing, gas clean up, and other failure mechanisms within the laser. Laser tubes of the prior art used epoxy to seal Brewster windows or mirrors. Power supplies used with these lasers were bulky, expensive, and employed designs which were not compatible with suppression of arcing within the laser tube.
For the reasons noted above, and in particular for use in the applications noted above, a need exists for lasers having one or more of reduced size, reduced weight, reduced power consumption, less restrictive cooing requirements, increased reliability, decreased cost of manufacture, and/or operation in combination with appropriate output wavelengths, appropriate instantaneous output power, and appropriate average output power.